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A gene expression screen identifies mRNAs that differ in abundance between two mRNA mixtures by a subtractive hybridization method. The two mRNA populations are converted to double-stranded cDNAs, fragmented, and ligated to linkers for polymerase chain reaction (PCR) amplification. The multiple cDNA fragments isolated from any given gene can be treated as alleles in a genetic screen. Probability analysis of the frequency with which multiple alleles are found provides an estimation of the total number of up- and down-regulated genes. We have applied this method to genes that are differentially expressed in amphibian tadpoletailtissue in the first 24 hr after thyroid hormone treatment, which ultimately induces tail resorption. We estimate that there are about 30 up-regulated genes; 16 have been isolated.
FIG. 1. Flow diagram for isolation of up-regulated genes. A plus
sign (+) refers to the mRNA isolated from tadpole tails treated with
thyroid hormone (3,3',5-triiodo-L-thyronine, T3) for 24 hr, as well as
the cDNAs derived from this + mRNA; - refers to mRNA and
cDNAs from untreated tadpoles. LH, long hybridization; SH, short
hybridization; BD, biotinylated driver DNA. The exact opposite
protocol is carried out simultaneously to obtain the down-regulated
genes.
FIG. 2. Enrichment of up-regulated genes (A), using + cDNA as
tracer and - cDNA as driver; and enrichment of down-regulated
genes (B), using - cDNA as tracer and + cDNA as driver. The
enrichment was assayed by PCR Southern analysis. The flow chart
(Fig. 1) and text detail each enrichment step. *, Up-regulated gene
6 (thyroid hormone receptorB); o, up-regulated gene 10; o, actin (22);
A, thyroid hormone receptor a; A, down-regulated gene 17. The
cDNA abundance is the number of molecules of each fragment for
every 500,000 cDNA molecules at each step of enrichment. Upper
arrow, minimum abundance of a specific DNA fragment required (in
probes) to generate hybridization signal for colonies that contain the
fragment in this screen; lower arrow, detection limit ofPCR Southern
blot analysis using a cloned probe.
Fig. 3. A comparison of differentially expressed genes in control
(-) and thyroid hormone-treated (+)-tadpoletail by Northern blot
and PCR Southern blot. About 10 pg of total RNA (left two lanes) or
1.5 ,ug of - or + cDNA (right two lanes) was loaded in each lane. (A)
Up-regulated gene 6 (thyroid hormone receptor P). (B) Downregulated
gene 17. (C) Xenopus actin (22).
FiG. 4. Poisson distribution analysis of the up-r mRNAs
identified by this gene expression screen. Th line shows the
theoretil Poisson distribution curve that-best fitsthe data points,
P'(n) P(n)N =JeAR-fnfN, where A = 0.8 and is theanumber of
non-cross-hybridizing f sbiated for any cDNA. By analogy
to a genetic screen isi the number of alles ond for each gene. P
is the probability in Poisson distribution, P(n) is a conversion ofP
by a constant N, the total number of esmated up-related ges,
such that P(n) equals the number of deret cDNAs that hive n
isolated non-ross-hybing . (pen circles represent the
number of isolated cDNAs in the gen expression screen with n
isolated "on-cross-hybridizing fragments. Where th t i
Poisson distribution curve meets the vertical axis anem of
the number of unidentified up-regulated cONAs genes) still present
in the library.
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